Cloning Of Thermophilic Protease PhpI And The Effect Of Mutant Y120P On Enzyme Properties

Proteases are a group of proteins that catalyze the cleavage of peptide bonds in peptides, polypeptides, and proteins using a hydrolysis reaction. From a biological standpoint, these enzymes are critical for cellular survival, particularly in removal of denatured proteins during stress events or of proteins that have completed their functions. Various proteases play distinct roles in the degradation of proteins, including protease that break down proteins and peptidases that break down the resulting oligopeptide products to single residues. So it is an important research direction of such enzymes. The cysteine protease PhpI (Pyrococcus horikoshii protease I) from the hyperthermophilic archaeon Pyrococcus horikoshii OT3 belongs to the PfpI family of DJ-1/ThiJ/PfpI superfamily, which is famous for the similarity of the 3D structures between members but with various functions. In order to understanding the structure and function of enzymes and the relationship of the catalytic mechanism, we utilized the moleculer technology and the method of protein engineering to study the biochemical characterization.The thermophilic protease gene PhpI has been successfully cloned and over expressed in soluble form. After the heat treatment and gel exclusion chromatography, the purified enzyme was in the form of dodecamer, which had been identified by Western Blotting and enzyme overlay analysis. Biochemical characterization revealed that the optimal reaction temperature and pH values were 80 oC and 8.0 respectively. It maintained relative activity above 80% in the range of pH 7.5-8.5. The protease PhpI was obviously inhibited by the cysteine protease inhibitors (e.g. DTT, Cys-HCl, 2-Me and IAA). It was likely that these inhibitors covalently modified the -SH of Cys100 and prevented the catalysis. The enzyme was strongly activated by 0.2%-5.0% Tween-20 and Triton X-100, while with high ability of organic reagent resistance. PhpI was highly inhibited by divalent metal ions, such as Zn2+, Cu2+, Fe3+, Ni2+, Co2+. In this study, we showed that PhpI was not only an aminopeptidase with broad specificity but also an endopeptidase preferred the arginine acid on the P1 site. The protease hydrolyzed the optimum synthetic substrate L-R-AMC with a kcat of 0.6348 min-1, a Km of 12μM and a kcat/Km of 0.052μM-1min-1 as an aminopeptidase. And the kinetic parameters of the best Endopeptidase substrate L-AAFR-AMC were as followed: kcat = 0.112 min-1, Km = 10μM, kcat/Km = 0.011μM-1min-1.Based on the 3D structure of PhpI, it was found that the large phenyl of Tyr 120 was at the entrance of the pocket, as a result that the diameter of the tunnel-like substrate binding pocket was about 8.2 ?. Meanwhile, there was a hydrogen bond between the carbonyl oxygen of Cys 100 and the nitrogen of Tyr 120. To investigate the function of Tyr 120 in the catalysis of PhpI, we designed and constructed the mutant Y120P in order to eliminate the side chain obstacle of Tyr 120 and to interrupt the hydrogen bond on the main chain.Compared to the parent enzyme, the properties of mutant Y120P had distinctly changed. Biochemical characterization showed that the best reaction temperature was 75 oC, about 5 oC lower than that of the PhpI, and it could keep higher relative activity between 30-70 oC. The results indicated that the substitution at the position might affect the thermo stability of the enzyme, resulting in the high hydrolysis activity in a larger range of temperature. Its optimum pH value was 7.5, which was 0.5 lower than parent enzyme and keeping relative activity above 80% in the range of pH 6.0 to 8.0; the pK1 of mutant Y120P decreased 1 point, which implied that the amino polarity at the position 120 might cause the changes of electrostatic environment at the activity center. The assay of the substrates specificity identified that the Y120P maintained the hydrolysis ability against the natural substrate gelatin, and increased the relative activity on the shorter peptide substrates. The kinetic results showed that the kcat cleaving L-R-AMC was 7-fold higher than that of wild type, which was 4.37 min-1, while holding similar Km. On the other hand, a 10-fold increase in kcat was observed on the hydrolysis of L-AAFR-AMC, it was also worthy noting that the Km value was only 1/2 compare with that of Wild type PhpI. The results above revealed that the major increase in the activities were due to the higher kcat values, indicating the Tyr->Pro mutagenesis mainly accellrated the catalysis of the enzyme but not substrate binding process. However, a 1/2 decrease in Km for L-AAFR-AMC also showed that this mutant was more favourable for bigger substrate binding. Molecular docking results also confirmed our deduction. Interupting the main chain hydrogen bond and eliminating the side chain benzyl of Tyr120 (Tyr->Pro) made it easier for substrate approach active site Cys100 and oxyanion hole Gly70, thereby increasing the enzyme catalysis. In the meanwhile, the mutant enlarged the binding entrance, thereby accelerating the substrate binding.In conclusion, we have successfully obtained the recombined thermophilic protease PhpI. Baced on the biochemical characterization and the analysis of the 3D structure, we identified the importance of the position120 which had a close relationship with the catalysis and the substrate specificity. Our study should be useful for the rational design of new enzymes.